The present invention relates to vaccine compositions for delivery to mucosal surfaces, and to a method of inducing, in a mammal, an immune response to an antigen or a mixture of antigens by delivering the antigen or mixture of antigens to a mucosal surface of the mammal.
More particularly, the present invention relates to vaccine compositions for inoculating a mammal such as a human against tetanus and B. pertussis infections.
It has long been the practice of clinicians to immunise human infants against a variety of common diseases by means of mixed vaccines which are directed against a plurality of diseases. For example, multiple-component vaccine compositions directed against diphtheria, tetanus and whooping cough have been available for a considerable number of years. Such vaccines have hitherto been administered by injection. The advantages of multiple-component vaccines are readily apparent in that the patient (usually an infant) is subjected to a much smaller number of potentially distressing injections than would otherwise be the case.
The majority of infectious diseases are initiated by contact with a mucosal surface. The infecting agent may remain at or within the mucous membranes during the course of the infection or may penetrate into the body and localise at other sites. The importance of the mucous membranes in the first line of defence against infectious disease can be gleaned from the fact that 90% of the lymphocytes of the body underlie such surfaces. Priming mucosal surfaces by immunisation so that they respond vigorously and effectively control pathogenic organisms they encounter would be advantageous. Unfortunately traditional immunisation regimes are ineffective at eliciting mucosal responses. The systemic and local (mucosal) immune systems appear to be compartmentalised and in general do not impinge on one another; that is parenteral immunisation with non-living vaccines stimulates mucosal immune responses weakly if at all. Mucosal immunisation (oral or intranasal) can evoke serum antibodies but this is usually less effective than parenteral immunisation. The immunocytes of the different mucous membranes form a vast intercommunicating network, termed the common mucosal immune system, such that topical immunisation of one surface (e.g. the gastrointestinal tract) may lead to an immune response at that surface and also distance surfaces such as the respiratory tract.
Manclark and Shahin (U.S. patent application Sre. No. 07/532,327, now abandoned, filed May 6, 1990xe2x80x94available through the US Department of Commerce, National Technical Information Service, Springfield, Va. 22161, U.S.A.)xe2x80x94have described the intranasal and intraduodenal administration of filamentous hemagglutinin (FHA) obtained from Bordetella pertussis and have illustrated that FHA is an effective mucosal immuogen. Manclark and Shahin speculated in U.S. Ser. No. 07/532,327, now abandoned, that the 69-kD outer membrane protein (P69) of B. pertussis would also be an effective mucosal immunogen, but presented no experimental data to show that this was the case.
The fact that there are very few mucosal vaccines commercially available indicates that there are problems with developing such vaccines Many non-living soluble antigens, particularly those used traditionally by immunologists, such as ovalbumin (OVA) and Keyhole Limpet Haemocyanin (KLH), are poor mucosal immunogens. Large doses of such antigens are necessary to induce any responses but large doses can also cause tolerance in the individual to subsequent parenteral exposure to antigen, a condition known as oral tolerance. Some microbial components such as the cholera toxin (CT) or E. coli heat-labile toxin (LT) or the non-toxic binding portions of these toxins (CT-B and LT-B) have been found to be potent mucosal immunogens eliciting strong secretary and circulating antibodies, but the reason why such molecules are good mucosal immunogens has not yet been fully elucidated. One property that may be important is the ability of these molecules to bind to mucosal epithelial cells via certain surface receptors, although it has been found in studies by others that there is not necessarily a correlation between the ability of an antigen to bind to eucaryotic cells and its mucosal immunogenicity.
Thus, as far as we are aware, there is currently no way of predicting with any certainty whether a given antigen will possess good mucosal immunogenicity.
We have now found that certain molecules make excellent mucosal immunogens and such components can be utilised in the development of a mucosally (intranasally or orally) delivered vaccine against the diseases whooping cough and tetanus. In particular, we have found that the P69 outer membrane protein (P69xe2x80x94also known as pertactin) from B. pertussis and the non-toxic immunogenic 50 Kd portion of tetanus toxin (C-Fragment) from C. tetanii are highly immunogenic when given intranasally. C-Fragment and P.69 were generated by DNA recombinant technology. Recombinant C-Fragment and P.69 produced from E. coli and yeast have been demonstrated to be immunogenic and protective in mice, see M. Roberts et al, Recombinant P.69/pertactin: imminogenicity and protection of mice against Bordetella pertussis infection; Vaccine 10, 43 (1992); and see also N. F. Fairweather et al, Infection and Immunity, 55, 2541 (1987).
In a first aspect, the invention provides the use of a mucosally immunogenically active substance comprising the 50 kD C fragment of tetanus toxin, an immunogenic fragment thereof, or a derivative thereof formed by amino acid deletion, substitution or insertion, for the manufacture of a vaccine composition for immunising a patient against tetanus infection.
In one particular embodiment of the invention, there is provided the use of a mixture of antigens for the manufacture of a vaccine composition for administration to a mucosal surface to induce an immune response in the mucosal surface against each of the said antigens, the mixture of antigens comprising:
(a) a mucosally immunogenically active substance comprising the 50 kD C fragment of tetanus toxin, an immunogenic fragment thereof, or a derivative thereof formed by amino acid deletion, substitution or insertion; and
(b) a mucosally immunogenically active substance comprising the P.69 outer membrane protein of B. pertussis;
an immunogenic fragment thereof, or a derivative thereof formed by amino acid deletion, substitution or insertion.
In a preferred embodiment the invention provides the use of a mixture of antigens as hereinbefore defined but wherein said mixture comprises in addition to (a) and (b);
(c)a mucosally immunogenically active substance comprising B. pertussis filamentous haemaglutinin, an immunogenic fragment thereof, or a derivative thereof formed by amino acid deletion, substitution or insertion.
In a further aspect, the invention provides a vaccine composition for application to a mucosal surface, the composition comprising antigen (a) or a mixture of antigens as hereinbefore defined and a pharmaceutically acceptable carrier.
In another aspect the invention, provides a method of immunising a host such as a mammal, (e.g. human) against infection, which method comprises administering an effective amount of antigen (a), or a mixture of antigens as hereinbefore defined, directly to a mucosal surface in the host to induce in said mucosal surface an immune response to each said antigen.
The mucosal delivery compositions of the present invention can be formulated, for example, for delivery to one or more of the oral, gastro-intestinal, and respiratory (e.g. nasal and bronchial) mucosa.
Where the composition is intended for delivery to the respiratory (e.g. nasal or bronchial) mucosa, typically it is formulated as an aqueous solution for administration as an aerosol or nasal drops, or as a dry powder, e.g. for inhalation.
Compositions for administration as nasal drops may contain one or more excipients of the type usually included in such compositions, for example preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents and the like.
The antigenic preparations of the present invention may also take the form of compositions intended to deliver the mixture of antigen to mucosal surfaces in the gastrointestinal tract. It is preferred that such compositions are provided with means for preventing degradation of the antigens by the gastric juices. For example, the compositions may take the form of capsules, e.g. microcapsules, in which the antigens are retained within a protective matrix or coating formed from an appropriate protective polymer such as a poly (glycolide), poly (lactide-co-glycolide), polyactyl starch, or pH-dependent coatings such as the polyacrylates or hydroxypropylmethyl cellulose phthalate.
The antigens may take the form of the tetanus toxin C fragment per se, the P.69 protein per se, or the B. pertussis haemaglutinin per se. Or it may take the form of a larger molecule containing one or more of the aforesaid antigens, immunogenically active fragments thereof, or derivatives formed by amino acid deletion, substitution and insertion, provided that the larger molecule is immunogenically active when administered directly to the mucosa.
The antigen or mixture of antigens typically is selected such that it is non-toxic to a recipient thereof at concentrations employed to elicit an immune response.
In one embodiment, two more of the antigens forming the mixture may be presented in a single molecule. Such a molecule may be prepared by recombinant methods by preparing a DNA construct containing genes coding for two or more of the antigens and expressing in a suitable host in accordance with known methods.
The individual antigenic substances making up the compositions of the invention may each also act as carriers for one or more other antigens. For example, an antigen such as the P.69 outer membrane protein or C-Fragment may be coupled to another antigen, and examples of such xe2x80x9cotherxe2x80x9d antigens include Haemophilus group B and meningococcal polysaccharide antigens.
In order to enhance the mucosal immunogenicity of the mixture of antigens or any component antigen thereof or appropriate immunogenic fragments thereof, they may be incorporated into appropriate carriers, for example virosomes, or the antigens or immunogenic fragments thereof may be expressed in suitable attenuated carrier strains of Salmonella. Immunogenicity may also be enhanced by incorporating appropriate mucosal adjuvants such as cholera toxin or E. coli heat-labile toxin, genetically detoxified variants thereof or their binding (B) sub-units in the vaccine.
The vaccine composition may optionally contain another mucosally immunogenically active portion of the tetanus toxin molecule. In addition, the mixed vaccine may contain one or more further mucosally immunogenically active antigens.
In one embodiment, the vaccine composition may, in addition to non-toxic immunogenic forms of tetanus toxin and pertussis antigens, contain non-toxic immunogenic forms of diphtheria toxin and immunogenic forms of Haemophilus influenzae group B polysaccharide (HiB), thereby providing a mucosal diphtheria-tetanus-pertussis (DTP) vaccine or DTPHiB vaccine.
The P.69 outer membrane protein of B. pertussis is a protein of approximately 51 KD molecular weight; see A. J. Makoff et al, xe2x80x9cProtective surface antigen P.69 of Bordetella pertussis: its characteristics and very high level expression in Escherichia colixe2x80x9d, Bio-Technology, 8, 1030 (1990).
It can be prepared and isolated according to the method disclosed in P. Novotny et al: The Journal of Infectious Diseases, 164, 114 (1991), or recombinant material prepared from E. coli by the method given in the article by A. J. Makoff et al referred to above. It can bind to eukaryotic cells.
Purified B. pertussis filamentous haemaglutinin usually contains polypeptides of differing molecular weight ranging from 98-220 KD, and can be isolated and purified from cell culture supernatants of B. pertussis, for example as described in the article by P. Novotny et al referred to above. The filamentous haemaglutinin is able to bind to eukaryotic cells and cause haemaglutination of sheep erythrocytes.
The C fragment of tetanus toxin is a peptide of approximately 50 KD molecular weight which can be isolated and purified form E. coli by the method described in A. J. Makoffet al., Bio/Technology, 7, 1043 (1989). The C fragment is characterised by an ability to bind the eukaryotic cells possessing the trisialoganglioside GT16 and by an ability to elicit protection in mice against lethal challenge with tetanus toxin.
The antigenic molecules of the present invention can be prepared by isolation and purification from the organisms in which they occur naturally, or they may be prepared by recombinant techniques and expressed in a suitable host such as E.coli in known manner. When prepared by a recombinant method or by synthesis, one or more insertions, deletions, inversions or substitutions of the amino acids constituting the peptide may be made. Each of the aforementioned antigens is preferably used in the substantially pure state. The quantity of the mixture of antigens administered will depend, in part, upon the purity of the individual antigens. Thus, for a substantially pure form of the P.69 outer membrane protein, a dose in the range from about 1-100 microgrammes/dose typically would be administered to a human, the actual amount depending on the immunogenicity of the preparation in humans when applied to mucosal surfaces.
For a substantially pure form of the B. pertussis filamentous haemaglutinin, and the 50 KD C fragment of tetanus toxin, a typical dose range would be of the order given above in respect of P.69 protein. In a typical immunisation regime employing the antigenic preparations of the present invention, the vaccine may be administered in several doses (e.g. 1-4), each dose containing 1-100 microgrammes of each antigen. The immunisation regime may involve immunisation purely by the mucosal route or by a combination of mucosal and parenteral immunisation. The dosage will in general depend upon the immunogenicity of the different antigens when applied to the respiratory or gastrointestinal tracts of humans.
The invention will now be illustrated in greater detail by reference to the specific embodiments described in the following examples.
The examples are intended to be purely illustrative of the invention and are not intended to limit its scope in any way.